Oral squamous cell carcinoma is a condition that will kill approximately half of patients afficated within five years of diagnosis and may leave surviving patients with severe esthetic and/or functional compromise. Oral cancer progresses through a series of morphologic changes, which if treated early, vastly improve prognosis. Through cancer is widely believed to be the result of chromosomal alterations leading to the activation of oncogenes and suppression of antioncogenes, the actual biochemical and molecular changes preceeding the morphologic progression of oral lesions are poorly understood. The principle aim of this work is the isolation of normal-specific genetic sequences that may be involved in the suppression of malignant phenotypes during oral carcinogenesis. To test that chromosomal rearrangements and deletions during oral cancer development result in the loss of genetic suppressors of malignant transformation, we used the well-established hamster cheek pouch carcinoma (HCPC) model. Normal and malignant oral keratinocytes from the cheek pouches of the Syrian hamster were cultured. Extensive phenotypic assays were performed to confirm the normal and malignant behaviors. Complementary DNA (cDNA) libraries have been constructed from the normal and malignant hamster oral keratinocytes representing expressed genes in each population. Genetic sequences that are uniquely and/or preferentially expressed in the normal hamster oral keratinocytes were isolated by the technique of subtractive hybridization. A subtraction library of the genetic clones isolated has been constructed. From this library, 130 candidates clones were identified as Type I (normal)-specific by differential colony hybridization. Southern blot analysis of these 130 subtraction clones demonstrated that 29 clones were Type I-specific. The normal-specific expression of 4 of 7 subtraction clones were confirmed by Northern blot analysis. Two of these clones demonstrated the loss of a genomic fragment upon Southern blot analysis. This finding is suggestive of a loss of heterozygosity, a common observation associated with several important tumor suppressor genes. Using a 5' and 3' RACE (rapid amplification of cDNA ends) approach, the full-length sequence of one of these subtraction clones has been isolated and determined to encode a novel 114 amino acid polypeptide. This subtraction clone was named doc, for deleted in oral cancer. Transfecting full-length doc into malignant hamster oral keratinocytes altered the behaviors of the recipients in terms of morphology, growth rate, and anchorage independent growth, suggesting reversion of the transformation phenotypes. This approach to the identification of tumor suppressor genes should hasten the identification as well as the understanding of tumor suppressors which are potentially critical to the biology, diagnosis and treatment of oral cancer. Key words: tumor suppressor genes, oral carcinogenesis, substractive hybridization